INORGANIC SELENIUM AND TELLURIUM SPECIATION IN AQUEOUS ...

51

Table XV

UV/Vis analysis of unamended cultures. Optical density of each fraction at 526 nm.

Fraction Replicate 1 Optical Density Average SD
0.0408 Replicate 2 Replicate 3 0.0309
1 0.0185 0.0506 0.0091
2 0.0012 0.0228 0.0292 0.0231 0.0446
3 0.0049 0.0319 0.1015 0.0048 0.0297
4 0.0695 0.0380 0.0024
5 0.0713 0.0111 0.0569 0.0575 0.0351
6 0.0527 0.0508 0.0122
7 0.1234 0.0024 0.0072 0.0897 0.0017
8 0.1279 0.1314 0.0314
9 0.1135 0.0002 0.0442 0.0902 0.0377
10 0.1089 0.0931 0.0232
11 0.1284 0.0481 0.053 0.1139 0.0193
12 0.1414 0.1089 0.0152
13 0.1803 0.0503 0.0495 0.1419 0.0282
14 0.0819 0.0704 0.0519
15 0.1378 0.0842 0.0614 0.1114 0.0273
16 0.128 0.0483 0.0661
17 0.0548 0.0956 0.1707 0.1154 0.0694
18 0.0488 0.0933 0.0525
19 0.2974 0.0672 0.0900 0.2753 0.0467
20 0.0012 0.0144 0.2456
21 0.0021 0.0715 0.0988 0.1754 0.0195
22 0.0291 0.0853 0.2138
23 0.0981 0.1152 0.0521

0.0931 0.0921

0.0828 0.1626

0.0901 0.0392

0.1603 0.0362

0.0014 0.0154

0.1464 0.145

0.0893 0.1419

0.5091 0.0194

0.0051 0.0368

0.4144 0.1098

0.0948 0.132

Optical density 52

1st layer (0.1 M sucrose)Replicate 1
2nd layer (1.5 M sucrose)Replicate 2
3rd layer (2.0 M sucrose)Replicate 3
0.5
4th layer (2.5 M sucrose)
0.4

0.3

0.2

0.1

0
0 5 10 15 20 25

Fraction Number

Figure 14. Tube profile showing the distribution of cells in each sucrose layer for
unamended bioreactor experiment.

53

Selecting a relevant reference sucrose solution
The concentration of sucrose in fractions from the sucrose density gradient is

different for each layer. This might effects the optical density at 526 nm. To check
whether there is any light scattering due to sucrose, optical density of each sucrose
solution was measured at 526 nm wavelength using deionized water as reference
solution. Data are shown in Table XVI.

Table XVI

Variation of optical density at 526 nm with the concentration of sucrose solutions.

Concentration of Sucrose Solutions (M) Optical Density at 526 nm
0.1 0.0106
1.5 0.3351
2.0 0.4046
2.5 0.4921

Proving Methods
Before analyzing biological samples, known concentrations of commercially

available tellurium and selenium standards were analyzed by hydride generation method
and inductively coupled plasma method respectively, to prove method validity. Observed
concentrations for 13.38 ppb Te samples are given in Table XVII and observed
concentrations for 600 ppb Se samples are given in Table XVIII. Percent recovery was
calculated by multiplying the ratio of observed average concentration/calculated
concentration (600 ppb) by 100.

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Table XVII

Observed concentrations for 13.38 ppb tellurium samples from HGAAS method.

Sample Number Obtained Concentration of tellurium (ppb)
1 11.90
2 11.54
3 12.58
4 17.18
5 17.18
6 17.19
14.59
Average 2.85
SD 109

% Recovery

Table XVIII

Observed concentrations for 600 ppb selenium samples from ICP-AES method.

Sample Obtained concentration of selenium (ppb)
Number
Se (0) Se (IV) Se (VI) Mixture
1 467.1
2 635.3 569.9 591.0 586.8
3 537.4 543.6 615.8 633.0
Average 546.6
SD 84.5 631.6 589.9 613.1
% Recovery 91.1
615.0 598.9 610.9

39.5 14.6 23.8

102.5 99.8 111.8

55

Control Experiments
To determine whether or not the hydride generation method absorbance is

produced only due to Te, the exact same HGAAS procedures were carried out for
samples from sterile culture medium without tellurium amendments. Data for this control
experiment are given in Table XIX.

Table XIX

Observed data for deionized water samples from HGAAS method.

Sample Number Concentration of tellurium (ppb)
1 1.45
2 1.41
3 1.52
4 1.34
5 1.28
6 0.22
1.20
Average 0.49
SD

56

CHAPTER IV
DISCUSSION AND CONCLUSIONS

ÒMany and varied techniques have been used in measuring selenium. They range
from original method of Robinson et al., 1934 who estimated the red color of elemental
selenium, to those based on spectrometry (including both the absorbance of light by
molecules in solution and atoms in a flame) to fluorescence methodsÓ (Muth et al., 1967).
Spectrometric techniques such as ultra violet-visible spectrometry, hydride generation
atomic absorption spectrometry, and inductively coupled plasma spectroscopy-atomic
emission spectroscopy were used to analyze cells, biologically produced tellurium and
selenium. The advantages of these methods over those before are higher accuracy, lower
standard deviations, and shorter analysis (sampling time is less than 1 min). Due to
instrument-specific software, data can be obtained directly (e.g. in HGAAS and ICP-AES
experiments concentration is given directly using calibration plots plotted by the
computer). Particularly for ICP-AES experiments, there is no need of converting
oxidation states of analytes, etc.

The only commercially available elemental form of tellurium is gray/black in color
and the available elemental form of selenium is black. After growing K27 bacteria reach
the stationary growth phase, the bioreactor amended with selenite becomes brick red in
color but the bioreactor amended with tellurate changes to gray/black in color. The reason
may be the great variety of allotropy for selenium and lower variety of allotropy for

57

tellurium but most probably the color of the stablest form of tellurium is gray/black. The
gray/black color is due to biologically produced Te0 and the brick red color is due to
biologically produced Se0 distributed throughout the bioreactor solution (Lortie et al.,
1992).

The great stability of the orange-red allotropic form of Se0 produced by the bacteria
or precipitated in a cell free medium obtained from a stationary-phase culture implies
that Se0 is tightly bound to some compound produced by the cells and is protected
from transformation into the black form (Kessi et al., 1999).

In 1997, Yu et al. observed that anaerobic, selenite-amended cultures of
Pseudomonas fluorescens K27 turned brick red, most likely from formation of red
elemental selenium, whereas selenate-amended cultures did not. The amounts of Se0
produced in these biological systems were at least 10-20 times larger in selenite-
compared to selenate-amended cultures for the same incubation time. So in this research
we used selenite amended cultures for our selenium amendment experiments to generate
the largest amounts of Se0. For tellurium experiments, cultures were amended with
tellurite because tellurite is less toxic than tellurate using the organismÕs specific growth
rate as a toxicity measure (Basnayake et al., 2001).

Tellurite amended bacterial cultures produced elemental Te and metallic tellurium
deposition inside cells (Taylor et al., 1988; Moore and Kaplan, 1992). Other researchers
have found evidence for the formation of elemental Se in
bacterial cultures amended with selenium salts (Levine, 1925; Tomei et al., 1995; Kessi
et al., 1999). When our tellurite amended bioreactor solution was centrifuged, a black
solid precipitated on the bottom of the polycarbonate tube and a yellow-colored

58

supernatant could be seen. The black solid contains elemental tellurium and cells and the
supernatant contains soluble Te species like tellurite. For selenite amended bioreactor
solution, the solid consists of elemental selenium and cells (brick-red color), and the
yellow supernatant consists of soluble Se species like selenite.

To prove the validity of the Te analytical method of metalloid determination,
samples of known (13.38 ppb) tellurium content were taken through all oxidation and
reduction reaction steps identically to biological samples (Table XVII) and these showed
recovery rates of 109% with a relatively small standard deviation (109% ± 2.85% n=6).
Samples from sterile culture medium without tellurium amendments also were treated
identically and showed an insignificant tellurium content of approximately 1 ppb (Table
XIX). The method we used for determination of tellurium is acceptable, although the
variance is high. I was unable to carry out a control experiment for Se since the minimum
detection limit for Se by inductively coupled plasma method was high (500 ppb); where
as the minimum detection limit for Te by hydride generation method was 3 ppb, as
determined by multiplying the concentration of blank given by the instrument, by a factor
of 3.

The tellurium experiments for four different bioreactor runs showed
approximately 66% of added tellurium was recovered in the liquid medium and 34% was
recovered in the solid phase. All the tellurium added to the bioreactor was collected and
analyzed and average recovery was approximately 100%. The standard deviation around
that mean was large (97% ± 13% n=4 bioreactor runs).

To prove the validity of the Se instrumental method, elemental selenium, selenite,
selenate and mixture of selenite and selenate samples of known selenium content were

59

prepared in sucrose medium with 10% nitric acid and were analyzed by ICP-AES. These
showed recovery rates between 91.1%-111.8% with a standard deviation between
14.6%-84.5% n=3 as shown in Table XVIII.

To find the concentration of Se0 inside the cells and outside the cells, we made an
effort to separate free Se0 from cells. Hence a sucrose density gradient separation
technique was performed according to the method described by Kessi et al., 1999.

Since biomass is a function of optical density and the highest optical density for
aqueous K27 cultures is given at 526 nm, absorbance of each fraction (from the sucrose
gradient) was measured at 526 nm wavelength to find the distribution of cells among the
layers (Stone et al., 1998). ICP-AES experiment was used to find the distribution of
selenium among the layers.

According to Figures 12 and 13, both cells and Se0 have penetrated to the bottom
sucrose layer. It could be seen by our naked eyes too, i.e. some tiny brick-red colored
particles and slimy colorless particles were sitting in the bottom sucrose layer in the
centrifuged sucrose density gradient. That means this sucrose density
gradient has not performed the desired separation since Se0 and cells ended up in the
same layer after centrifugation. Figure 12 further suggests that there is a little amount of
selenium in the other three layers. Those may be soluble selenium species such as
selenite left over from the decantation process. When the sucrose density gradient was
centrifuged, some cells were probably lysed due to high centrifugation speed. Maybe
because of these less dense cell particles, the optical density is shown in other sucrose
layers (Figure 13) too. Although the tube profiles in Figures 12 and 13 follow a similar
trend, there is a significant standard deviation in between replicate samples, in

60

determination of selenium as well as cells, especially for high concentrated sucrose
samples. Reasons for this high variance will be discussed later.

Since we did not get a good separation of cells and selenium, several different
sucrose density gradients were tried (e.g. sucrose layers of 0.15 M, 2.25 M, 2.73 M,
2.84 M). But their ability to separate these cells and Se0 were poorer than that of the
gradient discussed above (0.1 M, 1.5 M, 2 M, 2.5 M). These gradients also ended up
giving almost all Se0 and cells in one layer after the centrifugation.

Either all Se0 is bound to cells (in or/and on cells) or Se0 and cells have the same
density. If Se0 is bound to cells, a suggestion would be first the cells might be rinsed and
Se0 might in this way be taken off cells and then we might be able to analyze for free Se0
and Se0 with cells separately. If the Se0 and cells with Se0 are the same density then
another method such as electron microscopy might be use to determine Se content inside
cells.

The most widely used gradient material is sucrose due to its solubility,
transparency and cost. It is an almost chemically inactive material. Although sucrose is
readily available, it usually contains some trace UV absorbing material. The principal
problem with sucrose gradients is the high osmotic potential of concentrated solutions of
sucrose. The membrane around the cell, which is impermeable to sucrose, results in a
progressive loss of water from the cell as it moves into more concentrated sucrose layers.
The recommended maximum sucrose concentration is 65% (w/w) or 3.85 M (Price,
1927). Therefore in all of our trials the maximum concentration of sucrose of the bottom
layer was kept below 3.85 M. As shown in Table XVI, sucrose also scatters visible light.

61

Hence relevant sucrose solutions were used as references instead of using deionized
water.

One of the main problems in the determination of trace amounts of tellurium and
selenium in biological samples is the possibility of systematic errors due to the
difficulties associated with the complete mineralisation of organoselenium and
organotellurium compounds, that is, the difficulty of digesting biologically bound
metalloids to make them available for instrumental analysis (Sabe et al., 2001). The
factors that can be contributed to the high variance among the biological samples may be
other interfering metalloids or metals and variences in HGAAS procedures due to
unstable reagents, poorly optimized reduction steps, small errors in the pipetting reagents
involved that may have large consequences in ppb range, varying sample acidity, the
effect of nitric acid left over from the oxidation step on the reduction and hydride
generation step (Thompson et al., 1978; Sinemus et al., 1981; Hayrynen et al., 1985;
Dedina and Tsalev, 1995), crystallization of sucrose over time, and unwanted mixing of
sucrose layers at their interfaces. Finally inhomogeneous sampling procedures were a
concern. To minimize those kinds of effects, the following steps were taken: A small
linear working range (0-20 ppb) was chosen in the HGAAS method. Hydride generation
reagents (NaBH4 / NaOH) were freshly prepared daily. When preparing 0.35% NaBH4 /
0.5% NaOH solution for HGAAS reduction, NaBH4 was added to the NaOH solution
after dissolving all NaOH pellets. To decrease the effect of instrument drift, calibrations
were run between every 5 samples; samples were analyzed by HGAAS immediately after
chemical reduction, and the matrices of all samples and standards were made exactly the
same by controlling the acid concentration. When using background correction the

62

instrument gave higher % errors (25%) and poor R values (R=0.98) for Te samples with
known concentrations. But without background correction the % error was more
acceptable (9%) and the R value was very good (R=1.0000). For ICP-AES experiments,
calibration standards and samples had to be prepared freshly. The calibration standards
prepared one or more days before the analysis, gave poor R values (e.g. 0.81); whereas
freshly prepared calibration standards gave good R values (e.g. 0.9956 £ R £ 1).

Although our samples contained some metals other than tellurium or selenium
(trace elements from TSB medium and those contaminating our reagents), when the
samples were diluted into the linear working range these became low in concentration
(based on certified contaminations reported by manufacturers), below ranges that have
caused problems for others (Dedina and Tsalev, 1995; Narasaki, 1984). In HGAAS
experiments, concentration of residual nitric acid in the final samples was
also much below what has been reported to cause problems. To mix the bioreactor
solution well and to get a homogeneous mixture, the mixing speed was increased to 400
rpm from 200 rpm when sampling. Foaming prevented maintaining the mixing speed at
400 rpm throughout the entire bioreactor run.

The most important factors effecting error from the point of view of atomic
spectrometric techniques considered here are the density, viscosity, surface tension and
volatility of samples. Changes in viscosity and surface tension lead to changes in the
solution aspiration and nebulization, whereas density and volatility affect the aerosol
transport through the spray chamber of HGAAS or ICP-AES. For most inorganic acids
such as nitric or hydrochloric, the change in surface tension is small compared with the
changes viscosity and density. For this reason, it is expected that the drop size



64

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